Most of the plant species upon which Mankind is dependent for the bulk of its diet belong to that group of plants known collectively as the Gramineae. The Gramineae (Poaceae) are, from a commercial point of view, the most important family within the class of monocotyledonous plants. The Gramineae embrace, for example, the following subfamilies and genera:
______________________________________ Subfamily Genus within the subfamily ______________________________________ Bambusoideae Bamboo Andropogonoideae Saccharum [sugarcane] Sorghum Zea [corn] Arundineae Phragmites Oryzoideae Oryza [rice] Panicoideae Panicum (*) Pennisetum (*) Setaria (*) Pooideae (Festuciadeae) Poa (**) Festuca (**) Lolium (**) Bromus (**) Trisetum (**) Agrostis (**) Phleum (**) Dactylis (**) Alopecurus (**) Avena [oats] ( ) Triticum [wheat] ( ) Secale [rye] ( ) Hordeum [barley] ( ) Sorghum [tz,1/32 (*) (millets) (**) (grasses) ( ) (small grain cereals)
Among the subfamilies of the Gramineae the family Pooideae is a group of economically highly important plants that includes, for example, the two closely related subgroups consisting of the grasses and the small grain cereals.
Interestingly these Pooideae plants have also been the most difficult to manipulate scientifically. Until now, no generally applicable method is known for the regeneration of Pooideae plants, or fertile Pooideae plants, or for Pooideae plants containing stably incorporated exogenous DNA from protoplasts, although plant regeneration from cultured protoplasts is essential for the application of somatic hybridization and direct gene transfer. The present state of the art in gene transfer into cereals has recently been reviewed by Cocking, E. C., and Davey, M. R. [Science, 236 (1987) 1259-1262].
Sources for cereal cultures, protoplasts, the isolation of cereal protoplasts, and their properties are reported, for example, in the following book: ["Cereal Tissue and Cell Culture" Bright, S. W. J. and Jones, M. G. K., (eds) (1985) Nijhoff, M./Junk, W. Dr., Dordrecht].
Stable transformation has been already achieved in the Gramineae by chemically and electrically stimulated uptake of DNA into protoplasts ("direct gene transfer") [Potrykus, I., et al., Mol. Gen. Gent., 199 (1985) 183-188; Loerz, H., et al., Mol. Gen. Gent., 199 (1985) 178-182; Fromm, M. E., et al., Nature, 319 (1986) 791-793], but plant regeneration was not possible from the lines used in these studies.
So far, graminaceous plants have only been successfully regenerated from protoplasts other than of the subfamily Pooideae: For example, Abdullah, R., et al. [Bio/Technology, 4 (1986) 1087-1090] report the efficient plant regeneration from rice (subfamily: Oryzoideae) protoplasts through somatic embryogenesis. Yamada, Y., et al. [Plant Cell Reports, 5 (1986) 85-88] also describe rice plant regeneration from protoplast-derived calli. Also Rhodes, C., et al. [Biotechnology, 6 (1988) 56-60] describe the regeneration of non-fertile plants of maize. Cocking, E. C., and Davey, M. R. [supra] discuss the present state of the art in gene transfer in cereals.
The regeneration of graminaceous plants of the subfamily Pooideae from tissue cultures is known: Hanning, G. E., et al. [Theor. Appl. Genet., 63 (1982) 155-159] describe embryo and plantlet formation from leaf segment-derived callus of Dactylis glomerata L.
Some further examples of regeneration of Pooideae plants from cultured cells are reported in the following articles:
Lolium rigidum: Skene, K. G. M., et al., Zeitschr. Pflanzenz uchtung, 90 (1983) 130-135. PA0 Lolium perenne, Lolium multiflorum: Ahloowalia, B. S., Crop Science, 15 (1975) 449-452. PA0 Lolium multiflorum, Festuca arundinacea: Kasperbauer, M. J., et al., Crop Science, 19 (1979) 457-460. PA0 Alopecurus arundinaceus, Agropyron crystatum, Stipa viridula, Bromus inermis, Agropyron smithii: Lo, P. F., et al., Crop Science, 20 (1980) 363-367. PA0 Agrostis palustris: Krans, J. V., et al., Crop Science, 22 (1982) 1193-1197.
The state of tissue culture in forage grasses has also been reviewed by Ahloowalia, B. S. [Handbook of Plant Cell Culture, Ammirato et al. (eds), Macmillan, New York (1984) 91-125].
However, these Pooideae plants were not regenerated in these cases from the type of starting material described in the present application but from other types of cell cultures. It has not been demonstrated in the above examples that regeneration was de-novo by way of somatic embryogenesis. The above quoted references did not comprise the isolation and culture of protoplasts or the regeneration of plants from protoplasts.
Although there has been great interest in genetic transformation and regeneration of graminaceous plants of the subfamily Pooideae, there has been no description to date of a successful in-vitro method which can lead to regenerated, optionally transformed, protoplast-derived, plants or fertile plants (Cocking E. C. and Davey, M. R. [supra]).
Until now all investigations and every effort made in this direction failed, in so far as they resulted in embryos or at most in non-viable plantlets that died in an early stage and therefore could not be successfully transferred to soil [Ahloowalia, B. S. [Handbook of Plant Cell Culture, Ammirato et al. (eds), Macmillan, New York (1984) 91-125].
No description of a procedure for producing Pooideae protoplasts capable of undergoing differentiation to plants and whole fertile plants, much less of the regeneration of Pooideae plants from protoplasts or protoplast-derived calli, has appeared.